MadSci Network: Biochemistry

Re: How does one precisely identify proteins using mass spectrometry?

Date: Fri Mar 2 18:23:21 2007
Posted By: Luis Sojo, Faculty, Chemistry, Simon Fraser University
Area of science: Biochemistry
ID: 1172866932.Bc

Ritvik, That is a great question. The identificaiton of proteins by mass spectrometry (MS) is a bit complex. First, MS can provide you information on the molecular weight of the protein. Proteins acquire lots of protons (positive charges) during the ionization step prior to entering the mass spectrometer. This permits the detection of large proteins, but measuring mass is not enough, since there could be many proteins with very similar masses. In fact, since the masses can be quite large, a small difference in mass may not be detected by a particular mass spectrometer and we may therefore not be able to differentiate one protein from another.

In practice, what researchers do, is to digest the protein into peptides by using an enzyme such as trypsin. The resulting mixture of peptides is then analyzed by a liquid chromatograph coupled to a mass spectromter via an electrospray interface. This interface generate ions from the peptides allowing them to be detected by the mass spectrometer. The liquid chromaotraph permits us to separate the peptide mixture prior to its entering the mass spectrometer. The resulting data set is called a peptide map, which is a list of the peptides detected. A skillful mass spectroscopist can then identify the peptides and put together a map of the protein's primary structure or peptide composition and sequence. It is this sequence that is then used to identify the protein based on previously established sequences.

Hope this helps

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